The long-term goal of studies described in this application is to understand the diversification mechanisms which enable vertebrate organisms to synthesize antibodies specific for a seemingly unlimited variety of pathogens and other foreign substances. As a model system, the antibody response of inbred strain A mice to the cell wall carbohydrate (A CHO) of group A streptococci, the causative agent of strep throat, is being analyzed. The proposed research focuses on determining: (1) the number of distinct germ-line genes that encode anti-A CHO antibodies; (2) the contribution of combinatorial joining of genes to the diversity among anti-A CHO antibodies; (3) the extent to which somatic mutation contributes to diversity; (4) the influence of ontogeny, antigenic stimulation and isotype switching on diversification processes; and (5) structural correlates of antibody specificity and idiotypy. An estimate of the number of antibody variable region genes that encode anti-A CHO antibodies is obtained by comparative restriction mapping of DNA from anti-A CHO hybridomas utilizing Southern blot hybridizations with J gene probes to visualize rearranged, expressed genes. The initial data obtained with J probes will be verified with single-copy probes specific for the V gene of interest. The hybridomas are representative of primary and secondary antibody responses (antigenic stimulation), IgM and IgG3 producers (isotype switching), and antibody responses of young and adult mice (ontogeny). Using conventional molecular biological procedures, the variable region genes expressed by the hybridomas will be isolated and their nucleotide sequences determined. The corresponding germ-line genes will be isolated from embryonic DNA and sequenced. Analysis of these nucleotide sequences will allow a definitive assessment of mechanisms utilized in the diversification of anti-A CHO antibodies. Our approach to establishing structure-function correlates is to obtain hybridomas which express appropriate VH and VL genes, but fail to express functions associated with these genes, A CHO-binding and idiotypy. The nucleotide sequences of these "variant" V regions will be determined to identify "lesions" responsible for loss of function.